The present invention relates to a novel microbial process for producing L-ascorbic acid and D-erythorbic acid and salts thereof. More specifically, the present invention relates to a process for producing L-ascorbic acid or D-erythorbic acid from 2-keto-L-gulonic acid or 2-keto-D-gluconic acid, respectively, using a thermoacidophilic microorganism. The present invention also relates to a process for producing salts of L-ascorbic acid or D-erythorbic acid from salts of L-gulonic acid or 2-keto-D-gluconic acid, respectively, using a thermoacidophilic microorganism.
L-Ascorbic acid (vitamin C) is widely used in health care as well as in preparing food and animal feed, such as, for example, fish feed, and in cosmetics. D-Erythorbic acid is mainly used as an antioxidant for food additives.
L-Ascorbic acid has been produced from D-glucose by the well-known Reichstein method (Helv. Chim. Acta 17, 311-328, 1934). In this multi-step method, L-ascorbic acid is produced chemically from the intermediate 2-keto-L-gulonic acid. The method has been used commercially for more than 60 years, during which time many chemical and technical modifications have been made to improve the efficiency of the steps that produce the intermediates D-sorbitol, L-sorbose, diacetone-L-sorbose, diacetone-2-keto-L-gulonic acid, 2-keto-L-gulonic acid, and methyl 2-keto-L-gulonate, as well as improving the efficiency of the final product, L-ascorbic acid. The conversion of D-sorbitol to L-sorbose is the sole microbial step, the others being chemical steps. The conversion of diacetone-2-keto-L-gulonic acid into L-ascorbic acid has been performed by two different procedures: 1) deprotection to give 2-keto-L-gulonic acid, followed by esterification with methanol and base-catalyzed cyclization; and 2) acid-catalyzed cyclization to L-ascorbic acid directly from the protected or deprotected 2-keto-L-gulonic acid. These conversion processes must be performed in non-aqueous or low-aqueous reaction media. Environmentally and economically, carrying out the reaction in the absence of organic solvents is preferred.
D-erythorbic acid has been produced from D-glucose via 2-keto-D-gluconic acid. 2-keto-D-gluconic acid can be produced by fermentation using a strain belonging to the genus Pseudomonas, and via methyl 2-keto-D-gluconate.
Much time and effort has been devoted to finding other methods of producing L-ascorbic acid using microorganisms. Most studies on the microbial production of L-ascorbic acid have focused on the production of the intermediate 2-keto-L-gulonic acid, particularly from L-sorbose (G. Z. Yin et al., Sheng Wu Hsueh Pao. 20, 246-251, 1980; A. Fujiwara et al., European Patent Publication No.213 591; T. Hoshino et al., U.S. Pat. No. 4,960,695; and I. Nogami et al., European Patent Publication No. 221 707), from D-sorbitol (A. Fujiwara et al., European Patent Publication No. 213 591; T. Hoshino et al., U.S. Pat. No. 5,312,741; M. Niwa et al., W.I.P.O. Publication No. 95/23220; and S. F. Stoddard et al., WO 98/17819), or from D-glucose via 2,5-diketogluconic acid, with a single, mixed, or recombinant culture (T. Sonoyama et al., Appl. Environ. Microbiol. 43, 1064-1069, 1982; and S. Anderson et al., Science 230, 144-149, 1985). The 2-keto-L-gulonic acid can then be converted into L-ascorbic acid by chemical means, as described above.
The use of a biological process for the conversion of 2-keto-L-gulonic acid ester into L-ascorbic acid has recently been reported in Hubbs, U.S. Pat. No. 6,022,719 (xe2x80x9c""719 patentxe2x80x9d). This patent discloses a process for producing L-ascorbic acid by contacting 2-keto-L-gulonic acid, or an ester thereof, with a hydrolase enzyme catalyst, such as, for example, a protease, an esterase, a lipase, or an amidase. The ""719 patent discloses the formation of L-ascorbic acid from an ester of 2-keto-L-gulonic acid, such as, for example, butyl 2-keto-L-gulonate, but not the formation of L-ascorbic acid from 2-keto-L-gulonic acid itself. For example, it discloses that a Candida antartica B lipase catalyzed reaction formed 413 to 530 mg/l of methyl 2-keto-L-gulonate, but no L-ascorbic acid, from 1% (w/v) 2-keto-L-gulonic acid in the presence of 8.6% methanol, at a pH of from 3.1 to 3.2, at 38xc2x0 C. The ester synthetic activity of Candida antartica B lipase on 2-keto-L-gulonic acid, an xcex1-keto-carboxylic acid, at an acidic pH, was apparently positive. However, intramolecular ester formation by this lipase was negligible.
In addition to the hydrolase reaction, ester bond synthesis reactions, such as those used for the formation of proteins (amino-esters), fatty acid esters (carboxyl-esters), and nucleotide chains (phospho-esters), are all highly functional in cells. Even in the aqueous phase, these ester bond synthesis reactions proceed unidirectionally, and are seldom inhibited by the product, particularly when compared with the reverse reaction of a hydrolase. These reaction systems require a supply of activated esters, such as, for example, activated transfer ribonucleic acid (tRNA), adenosine triphosphate (ATP), acyl coenzyme A (acyl-CoA), and the like, which are generated by energy-converting metabolism in cells. The xe2x80x9cin vitroxe2x80x9d reconstitution of these reactions requires a stoichiometric supply, or a regeneration system, of energy donors (e.g., ATP). Such energy donors are expensive to use in the commercial production of vitamins, as well as other chemicals, such as L-ascorbic acid and D-erythorbic acid. Thus, the utilization of intact cells is one of the more preferred commercial methods.
The chemical conversion of 2-keto-L-gulonic acid to L-ascorbic acid via 2-keto-L-gulonic acid xcex3-lactone is an acid-catalyzed reaction accompanied by the elimination of a water molecule. The principle step in the reaction is a carboxyl ester bond formation to form a xcex3-lactone ring in a 2-keto-L-gulonic acid molecule. Therefore, especially in the aqueous phase, the final state of the equilibrium reaction is determined by physico-chemical conditions. The productivity of L-ascorbic acid from 2-keto-L-gulonic acid by chemical conversion is considerable, even in the aqueous phase, but it is not sufficient for commercial application. However, performing the process in the aqueous phase, or in an aqueous phase with a low content of an organic solvent, is highly desirable due to its cost effectiveness, and for complying with environmental demands. Accordingly, the biological enhancement of the chemical conversion would be desirable for production in the aqueous phase.
Both high temperature and acidic (i.e., low) pH are desirable reaction parameters for improving the efficiency of the chemical reaction. However, in general, such physico-chemical conditions are known to be biologically incompatible with the cell survival and/or cellular activity of most microorganisms viable under mesophilic conditions. The utilization of thermophilic or acidophilic microorganisms is well known. However, there have been few examples using thermoacidophilic microorganisms which have tolerance to both heat and acid.
It has now been found that the conversion of 2-keto-L-gulonic acid, as the free acid or as its sodium potassium or calcium salt, to L-ascorbic acid, or the respective salt, in the aqueous phase, can be directly and favorably performed by thermoacidophilic microorganisms under biologically extreme conditions, such as, for example, at high temperature and low (i.e., acidic) pH. It has further been found that the conversion of 2-keto-D-gluconic acid, as the free acid or as its sodium, potassium or calcium salt, to D-erythorbic acid, or the respective salt, in the aqueous phase, can also be directly and favorably performed by thermoacidophilic microorganisms under biologically extreme conditions.
One embodiment of the present invention is a process for producing L-ascorbic acid, or a sodium, potassium or calcium salt thereof from 2-keto-L-gulonic acid, or a sodium, potassium or calcium salt of 2-keto-L-gulonic acid involving: incubating in a solution a substrate having 2-keto-L-gulonic acid as a free acid or as a sodium, potassium or calcium salt of 2-keto-L-gulonic acid, and a thermoacidophilic microorganism at about 30xc2x0 C. to about 100xc2x0 C. and at a pH from about 1 to about 6 to form L-ascorbic acid or a salt thereof; and isolating the L-ascorbic acid or salt thereof from the microorganism or the solution.
Another embodiment of the present invention is a process for producing D-erythorbic acid, or a sodium, potassium or calcium salt thereof from 2-keto-D-gluconic acid or a sodium, potassium or calcium salt of 2-keto-D-gluconic acid involving: incubating in a solution a substrate comprising 2-keto-D-gluconic acid as a free acid or as a sodium, potassium or calcium salt of 2-keto-D-gluconic acid, and a thermoacidophilic microorganism at about 30xc2x0 C. to about 100xc2x0 C. and at a pH from about 1 to about 6 to form D-erythorbic acid or a salt thereof; and isolating the D-erythorbic acid or salt thereof from the microorganism or the solution.
Another embodiment of the present invention is an isolated microorganism selected from the group consisting of Alicyclobacillus sp. NA-20 (DSM No. 13649), Alicyclobacillus sp. NA-21 (DSM No. 13650), and Alicyclobacillus sp. FJ-21 (DSM No. 13651).
A further embodiment of the present invention is a process for producing L-ascorbic acid or a salt thereof from 2-keto-L-gulonic acid or a salt thereof involving:
(a) contacting 2-keto-L-gulonic acid with a microorganism selected from the group consisting of Alicyclobacillus sp. NA-20 (DSM No. 13649), Alicyclobacillus sp. NA-21 (DSM No. 13650), and Alicyclobacillus sp. FJ-21 (DSM No. 13651) in a culture medium sufficient to support the growth of the microorganism under the following conditions:
(i) a temperature of about 30xc2x0 C. to about 100xc2x0 C.; and
(ii) a pH from about 1 to about 6; and
(b) isolating the L-ascorbic acid or a salt thereof from the microorganism or the medium.
Another embodiment of the present invention is a process for producing D-erythorbic acid, or a salt thereof from 2-keto-D-gluconic acid or a salt thereof involving:
(a) contacting 2-keto-D-gluconic acid with a microorganism selected from the group consisting of Alicyclobacillus sp. NA-20 (DSM No. 13649), Alicyclobacillus sp. NA-21 (DSM No. 13650), and Alicyclobacillus sp. FJ-21 (DSM No. 13651) in a culture medium sufficient to support the growth of the microorganism under the following conditions:
(i) a temperature of about 30xc2x0 C. to about 100xc2x0 C.; and
(ii) a pH from about 1 to about 6; and
(b) isolating the D-erythorbic acid or a salt thereof from the microorganism or the medium.
A further embodiment of the present invention is a microorganism that produces L-ascorbic acid or a salt thereof or D-erythorbic acid or a salt thereof having the following characteristics:
(a) an rDNA sequence that is at least 98.1% identical to SEQ ID NO: 1, 2 or 3 using the Genetyx-SV/R software program;
(b) a rod-shaped morphology;
(c) a width of about 0.8 xcexcm;
(d) an inability to grow under anaerobic conditions;
(e) exhibiting catalase activity;
(f) {overscore (xcfx89)}-Cycohexylic acid as its major fatty acid;
(g) an ability to grow at a pH of 3.0 and a temperature of 60xc2x0 C.;
(h) an inability to grow under the following conditions:
(i) an ability to produce a (1) L-ascorbic acid or a salt thereof from 2-keto-L-gulonic acid or a salt thereof, (2) D-erythorbic acid or a salt thereof from 2-keto-D-gluconic acid or a salt thereof, or (3) both L-ascorbic acid or a salt thereof and D-erythorbic acid or a salt thereof from 2-keto-L-gulonic acid or a salt thereof and 2-keto-D-gluconic acid or a salt thereof, respectively.
The process of the present invention involves incubating 2-keto-L-gulonic acid or 2-keto-D-gluconic acid, each as the free acid, or as its sodium, potassium, or calcium salt, and cells of a thermoacidophilic microorganism capable of producing and/or enhancing the production of L-ascorbic acid, or its sodium, potassium, or calcium salt, from 2-keto-L-gulonic acid, or its sodium, potassium, or calcium salt, or D-erythorbic acid, or its sodium, potassium, or calcium salt, from 2-keto-D-gluconic acid, or its sodium, potassium or calcium salt, at a high temperature (i.e. at temperatures from about 30xc2x0 C. to about 100xc2x0 C.) and at an acidic pH (i.e. at a pH from about 1 to about 6), in a solution as shown in Example 2, hereinafter, and isolating the L-ascorbic acid, or its sodium, potassium, or calcium salt, or D-erythorbic acid, or its sodium, potassium, or calcium salt, from the solution.
As used herein, xe2x80x9cor its sodium, potassium or calcium saltxe2x80x9d or an equivalent expression as applied to xe2x80x9c2-keto-L-gulonic acidxe2x80x9d, xe2x80x9c2-keto-D-gluconic acidxe2x80x9d, xe2x80x9cL-ascorbic acidxe2x80x9d, or xe2x80x9cD-erythorbic acidxe2x80x9d will be referred to hereinafter as xe2x80x9cor its saltxe2x80x9d. Moreover, any given concentrations of these acids or their salt forms will be expressed as being based on the free acid form even though a salt form may be present, unless clearly stated for the particular acid, or the respective salt form that is present.
As used herein, a xe2x80x9cthermophilic microorganismxe2x80x9d is a microorganism with optimal growth at a temperature above about 55xc2x0 C. As used herein, an xe2x80x9cacidophilic microorganismxe2x80x9d is a microorganism with optimal growth at a pH in the acidic range, preferably below about pH 6, and no growth at a pH in the neutral range (i.e., in the pH range from about 6 to about 8). Thus, a xe2x80x9cthermoacidophilic microorganismxe2x80x9d is a microorganism with both of these properties, i.e., optimal growth at a temperature above about 55xc2x0 C., and at a pH below about 6, and no growth in the pH range from about 6 to about 8. The term xe2x80x9cthermoacidophilic microorganismxe2x80x9d, as used herein, also includes mutants of a thermoacidophilic microorganism, which also have the above-defined temperature and pH growth characteristics.
The term xe2x80x9cgrowthxe2x80x9d as used in the present invention means that a colony formation can be observed after 20 hours of incubation. The term xe2x80x9cno growthxe2x80x9d as used in the present invention means that no colonies are observed after incubation for 20 hours.
Normally, thermoacidophilic microorganisms can be prokaryotes, or can be isolated from prokaryotes, and are classified under both Archaea and Bacteria. In the Archaea domain, the genera Sulfolobus (T. D. Brock et al., Arch. Mikrobiol. 84, 54-68, 1972) and Thermoplasma (M. DeRosa et al., Phytochemistry 170, 1416-1418, 1970), are well-known thermoacidophilic microorganisms. The genera Acidanus (A. H. Segerer et al., Int. J. Syst. Bacteriol. 36, 559-564, 1986), Desulfurolobus (W. Zilling et al., Syst. Appl. Microbiol. 8, 197-209, 1986), Metallosphaera (G. Huber et al., Syst. Appl. Microbiol. 12, 38-47, 1989), Picrophilus (C. Schleper et al., J. Bacteriol. 177, 7050-7059, 1995), and Stygiolobus (A. H. Segerer et al., Int. J. Syst. Bacteriol. 41, 495-501, 1991), have also been reported as being thermoacidophilic microorganisms of the Archaea domain. In the Bacteria domain, the genera Acidimicrobium (D. A. Clark et al., Microbiology 142, 785-790, 1996), Acidothermus (F. Rainey et al., FEMS Microbiol. Lett., 108, 27-30, 1993), Sulfobacillus (R. S. Golovacheva et al., Microbiology 47, 658-665, 1978) and Alicyclobacillus (G. Darland et al. J. Gen. Microbiol. 67, 9-15, 1971; G. Deinhard et al., Syst. Appl. Microbiol. 10, 47-53, 1987) are thermoacidophilic microorganisms.
Thermoacidophilic microorganisms that can be used in the present invention include any thermoacidophilic microorganism which is capable of producing and/or enhancing the production of L-ascorbic acid, or its salt, from 2-keto-L-gulonic acid, or its salt, or the production of D-erythorbic acid, or its salt, from 2-keto-D-gluconic acid, or its salt.
The thermoacidophilic microorganisms used in the present invention can be obtained from any kind of natural source, such as, for example, soils and hot spring water, as well as from artificial sources, such as, for example, processed acidic foods and beverages (e.g., fruit juices and mixed fruit/vegetable juices).
The more extreme the conditions (ie., the higher the temperature and the lower (i.e., more acidic) the pH) under which any particular thermoacidophilic microorganism displays tolerance, the more preferably this microorganism is used in the process of the present invention. Besides tolerance to heat and acidity, thermoacidophilic microorganisms which are also tolerant to a high concentration (i.e., from about 5% to about 20% (w/v)) of 2-keto-L-gulonic acid or its salt, or of 2-keto-D-gluconic acid, or its salt, in solution, when incubated at high temperature and acidic pH, are also preferably used in the process. In addition, thermoacidophilic microorganisms with the aerobic and chemoorganotrophic characteristics described herein are preferred for the efficient (i.e., rapid) production of cells.
Preferred thermoacidophilic microorganisms are those derived from prokaryotes, including bacteria and archaea. More preferred microorganisms are thermoacidophilic bacteria. Especially preferred thermoacidophilic microorganisms are bacteria belonging to the genus Alicyclobacillus.
Among thermoacidophilic bacteria, the genus Alicyclobacillus embraces most of the strictly aerobic, spore-forming, rod-shaped and chemoorganotrophic bacteria. These microorganisms were initially assigned to the genus Bacillus. However, phylogenetic analysis based on sequence comparisons of the 16S rRNA gene has shown that the genus Alicyclobacillus belongs to a distinct line of descent within the low G+C Gram-positive lineage of Bacillus (J. D. Wisotzkey et al., Int. J. Syst. Microbiol. 42, 263-269, 1992). The three validly taxonomically described species of the genus Alicyclobacillus (A.) are: A. acidocaldarius (DSM 446T, G. Darland et al., J. Gen. Microbiol. 67, 9-15, 1971), A. acidoterrestris (DSM 3922T, G. Deinhard et al., Syst. Appl. Microbiol. 10, 47-53, 1987) and A. cycloheptanicus (DSM 4006T, G. Deinhard et al., Syst. Appl. Microbiol. 10, 68-73, 1987). Besides sequence comparisons of the 16S rRNA genes, the most distinguishable characteristic of these microorganisms is the presence of structural units of xcfx89-cyclohexyl fatty acids (xcfx89-cyclohexylundecanoic acid, xcfx89-cyclohexyltridecanoic acid) or of xcfx89-cycloheptyl fatty acids (xcfx89-cycloheptylundecanoic acid, xcfx89-cycloheptyltridecanoic acid) in their cellular membranes (L. Albuquerque et al., Int. J. Syst. Evol. Microbiol. 50, 451-457, 2000). Several strains with the characteristics of the genus Alicyclobacillus have been isolated so far from acidic soils within geothermal areas and from certain non-geothermal soils. In addition to soil samples, they have also been isolated from many acidic beverages as spoilage bacteria (G. Cerny et al., Z Lebens Unters Forsch 179, 224-227, 1984; K. Yamazaki et al., Biosci. Biotech. Biochem. 60, 543-545, 1996; M. Niwa et al., Japanese Patent Publication (Kokai) No. 140696/1996). Recently, in addition to the three validly named species, a wide diversity of genospecies among the genus Alicyclobacillus have been proposed. (A. Hiraishi et al., J. Gen. Appl. Microbiol. 43, 295-304, 1997; L. Albuquerque et al., Int. J. Syst. Evol. Microbiol. 50, 451-457, 2000).
Preferred thermoacidophilic microorganisms used in the present invention have the following characteristics:
1) Thermoacidophilic growth:
Showing growth at pH 3.0 at 60xc2x0 C. in 20 hours, but showing no growth at pH 3.0 at 30xc2x0 C., or at pH 6.5 at 30xc2x0 C., or at pH 6.5 at 60xc2x0 C., in 20 hours.
2) xcfx89-cyclohexyl fatty acids:
Having xcfx89-cyclohexyl fatty acid structural units in their cellular membranes according to gas chromatography-mass spectrometry (GC/MS) analysis.
3) 16S rRNA sequence similarity:
Phylogenic analysis of 16S genes coding for rRNA sequences confirms the allocation to the genus Alicyclobacillus.
The thermoacidophilic microorganisms used in the present invention can be obtained from natural and artificial sources, as indicated above, or commercially from culture depositories. For isolating the microorganisms from natural and artificial sources, the appropriate microorganism source, such as, for example, a natural source soil or hot spring water, or an artificial source, such as, for example, processed acidic food or beverage, is preferably cultured in an aqueous medium and/or on a solid medium, supplemented with appropriate nutrients under aerobic conditions. The cultivation is preferably conducted at temperatures above about 40xc2x0 C. and at a pH below about 5, more preferably above about 50xc2x0 C. and below about pH 4, and most preferably above about 55xc2x0 C. and below about pH 3.5. While the cultivation period varies depending upon the pH, temperature, and nutrient medium used, a period of 12 hours to several days will generally give favorable results.
Thermoacidophilic microorganisms belonging to the genus Alicyclobacillus and which are most preferably used in the present invention are Alicyclobacillus sp. DSM No. 13652 and DSM No. 13653, which can be obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Mascheroder Weg 1b, D-38124 Braunschweig, Germany, and the new strains, Alicyclobacillus sp. NA-20 and NA-21, which were isolated from a soil sample collected at Iwate Prefecture, Japan, and Alicyclobacillus sp. FJ-21, which was isolated from a commercial acidic beverage (i.e., fruit juice) purchased at Kamakura-shi, Kanagawa Prefecture, Japan.
All five of these thermoacidophilic microorganisms were deposited under the Budapest Treaty on Aug. 16, 2000 at the DSM and were allotted the following accession numbers:
Alicyclobacillus sp. DSM No. 13652
Alicyclobacillus sp. DSM No. 13653
Alicyclobacillus sp. NA-20: DSM No. 13649
Alicyclobacillus sp. NA-21: DSM No. 13650
Alicyclobacillus sp. FJ-21: DSM No. 13651
As indicated above, mutants of the above mentioned thermoacidophilic microorganisms can also be used. A mutant of a microorganism according to the present invention may be obtained by treating a wild type strain with a mutagen, such as, for example, by irradiation with ultraviolet rays, X-rays, xcex3-rays, or by contact with nitrous acid or other suitable mutagens. A mutant may also be obtained by isolating a clone occurring by spontaneous mutation, which may be effected using methods known by skilled artisans for such purposes. Many of these methods have been described in specialized publications, such as, for example, xe2x80x9cChemical Mutagensxe2x80x9d edited by Y. Tajima, T. Yoshida and T. Kada, Kodansha Scientific Inc., Tokyo, Japan, 1973.
As used herein, a xe2x80x9cmutantxe2x80x9d is any microorganism that contains a non-native polynucleotide sequence or a polynucleotide sequence that has been altered from its native form (such as, for example, by rearrangement or deletion or substitution of from 1-100, preferably 20-50, more preferably less than 10 nucleotides). As noted above, such a non-native sequence may be obtained by random mutagenesis, chemical mutagenesis, UV-irradiation, and the like. Preferably, the mutation results in the increased production (compared to a non-mutant parental strain using the assay procedures set forth in the Examples) of L-ascorbic acid, D-erythorbic acid, salts of L-ascorbic acid and D-erythorbic acid, and combinations thereof. Methods for generating, screening for, and identifying such mutant cells are well known in the art.
Moreover, biologically and taxonomically homogeneous cultures of Alicyclobacillus sp. DSM No. 13652, DSM No. 13653, NA-20 (DSM No. 13649), NA-21 (DSM No. 13650), or FJ-21 (DSM No. 13651), can be used. As used herein, xe2x80x9cbiologically and taxonomically homogeneous culturesxe2x80x9d are cultures showing the following biological and taxonomical characteristics:
growth: aerobic and thermoacidophilic
spore: forming
cell morphology: rod-shaped
major fatty acids: xcfx89-cyclohexyl fatty acids
phylogenetical position: closest (i.e., more than 90% identity in the nucleotide sequence of 16S rRNA gene) to the strains classified in the genus Alicyclobacillus, such as, for example, Alicyclobacillus sp. DSM No. 13652, A. sp. UZ-1, A. sp. MIH-2, A. sp. KHA-31 and A. acidocaldarius DSM446T, whereby the identity in the nucleotide sequence is defined using the Nucleotide Sequence Homology program (Genetyx-SV/R, version 4.0, Software Development Co., Tokyo, Japan) with default conditions (unit size to compare=1)
The above mentioned thermoacidophilic microorganisms can be used in any form, preferably as intact cells, modified cells, or immobilized cells. Methods for immobilizing cells are well known in the art (see, for example, W. M. Fogarty et al., Microbial Enzymes and Biotechnology, 2nd Edition, Elsevier Applied Science, pp. 373-394 (1983), and Japanese Patent Publication No. 61265/1994).
Thermoacidophilic microorganisms can be screened to assess their suitability for use in the process of the present invention by the following method:
The appropriate microorganism source is cultured in an aqueous medium containing the substrate 2-keto-L-gulonic acid, or its salt, or 2-keto-D-gluconic acid, or its salt, and supplemented with appropriate nutrients under moderately aerobic conditions (i.e., under aerobic incubation without enforced aeration or vigorous agitation). The concentration of the substrate, 2-keto-L-gulonic acid, or its salt, or 2-keto-D-gluconic acid, or its salt, for carrying out the cultivation may be from about 3% (w/v) to about 20% (w/v), preferably from about 4% (w/v) to about 18% (w/v), and more preferably from about 5% (w/v) to about 16% (w/v). The incubation may be conducted at pHs from about 0.5 to about 4.0, preferably from about 1.0 to about 3.5, and more preferably from about 1.5 to about 3.0, and at temperatures from about 45xc2x0 C. to about 90xc2x0 C., preferably from about 50xc2x0 C. to about 85xc2x0 C., and more preferably from about 55xc2x0 C. to about 80xc2x0 C. While the incubation period varies depending upon the pH, temperature, and nutrient medium used, a period of about 12 hours to several days will generally give favorable results. After the incubation, the suitability of the screened thermoacidophilic microorganism in the process of the present invention may be assessed by its degree of productivity, (i.e., product accumulation) of L-ascorbic acid, or its salt, or D-erythorbic acid, or its salt, compared with the productivity in a xe2x80x9cblankxe2x80x9d incubation (i.e. without the thermoacidophilic microorganism), whereas an over 2-fold increase of productivity over the productivity in a xe2x80x9cblankxe2x80x9d incubation is preferred.
In the incubation described above, the presence of a high concentration of the substrate, 2-keto-L-gulonic acid, or its salt, or 2-keto-D-gluconic acid, or its salt, in addition to a high temperature and an acidic pH, may present extreme physico-chemical conditions even for a thermoacidophilic microorganism. The tolerance to 2-keto-L-gulonic acid, or its salt, or 2-keto-D-gluconic acid, or its salt, at high temperature and acidic pH is an important characteristic of the thermoacidophilic microorganisms used in the process of the present invention for maintaining cell viability.
The incubation for producing, and/or enhancing the production of, L-ascorbic acid, or its salt, from 2-keto-L-gulonic acid, or its salt, or D-erythorbic acid, or its salt, from 2-keto-D-gluconic acid, or its salt, with cells of a thermoacidophilic microorganism in the process of the present invention is effected in an aqueous phase solution. The solvent for the aqueous phase is preferably water alone (i.e., without any added other solvent(s)). If an additional solvent is used, however, a lower alkanol such as methanol is preferred.
The incubation for producing, and/or enhancing the production of, L-ascorbic acid from 2-keto-L-gulonic acid, or D-erythorbic acid from 2-keto-D-gluconic acid, each of these products or substrates being present as the free acid or the respective sodium, potassium, or calcium salt, requires nutrients, such as, for example, assimilable carbon sources, digestible or assimilable nitrogen sources, and inorganic substances, trace elements, vitamins, L-amino acids, and other growth promoting factors. As assimilable carbon sources, D-glucose, sucrose, D-glucono-xcex4-lactone, starch and the like can be employed. Various organic or inorganic substances may be employed as nitrogen sources, such as, for example, yeast extract, meat extract, peptone, casein, corn steep liquor, urea, amino acids, nitrates, ammonium salts, such as, for example, ammonium sulfate, and the like. As inorganic substances, magnesium sulfate, potassium phosphate, sodium chloride, potassium chloride, calcium chloride, and the like, may be employed. Furthermore, as trace elements, sulfates) hydrochlorides or phosphates of calcium, magnesium, zinc, manganese, cobalt and iron, may be employed. Preferred as inorganic salts are monopotassium phosphate, magnesium sulfate, ferrous sulfate and manganese sulfate. If necessary, conventional nutrient factors, or an antifoaming agent, such as, for example, animal oil, vegetable oil or mineral oil, can be added.
The conditions of the incubation may vary depending on the species and genetic character of the thermoacidophilic microorganism employed. The incubation is effected at what is considered to be a high temperature for an incubation (i.e., at temperatures from about 30xc2x0 C. to about 100xc2x0 C., preferably from about 40xc2x0 C. to about 95xc2x0 C., most preferably from about 55xc2x0 C. to about 95xc2x0 C.), at an acidic pH (i.e., at a pH from about 1.0 to about 6.0, preferably from about 1.0 to about 4.5, most preferably from about 1.5 to about 3.0), under aerobic conditions. Normally, an incubation period ranging from about 1 to about 100 hours is sufficient.
The suitable initial concentration of 2-keto-L-gulonic acid, or its salt, or of 2-keto-D-gluconic acid, or its salt, for the incubation depends on the particular thermoacidophilic microorganism used. However, a concentration of 2-keto-L-gulonic acid, or its salt, or of 2-keto-D-gluconic acid, or its salt, from about 5% (w/v) to about 20% (w/v), preferably from about 10% (w/v) to about 15% (w/v), based on the (equivalent) amount of free acid, is generally used.
The process of the present invention shows the following characteristics:
a) Specific production rate of L-ascorbic acid or its salt:
The specific production rate for L-ascorbic acid is, for example (in the presence of 8% (w/v) 2-keto-L-gulonic acid, the substrate, and 2.5 g/L L-ascorbic acid, the product, at 59xc2x0 C. and pH 2.5, for 20 hours by the strain NA-21 (DSM No. 13650)) about 2.3 mg of L-ascorbic acid/mg of crude cellular protein/hour. This is based on the results given in Example 7, hereinafter.
b) Product inhibition:
The production, in the process of the present invention, is seldom inhibited by the product, L-ascorbic acid, or its salt, or D-erythorbic acid, or its salt. In addition, the process of the present invention may provide higher conversion yields than those obtained by reversible reactions in the aqueous phase.
The L-ascorbic acid, or its salt, or D-erythorbic acid, or its salt, formed in solution may be isolated, i.e., separated and/or purified, by conventional methods known in the art. The respective products may be isolated from the medium or the cells depending on the circumstances. If the product is the sodium, potassium or calcium salt of the respective acid, this salt may, if desired, be converted into the respective free acid by conventional methods known in the art. In each case, isolation of the product may be effected by methods relying upon the differences in properties between the product and impurities (including the non-converted substrate), such as, for example, solubility, adsorbability, electrochemical properties, and the distribution coefficient between two solvents. The use of an absorbent, such as, for example, an ion exchange resin, is a convenient method for isolating the product. An electro-dialysis system is another convenient method for isolating the product. If the product is insufficiently pure for its subsequent use, it may be purified by conventional methods, such as, for example, recrystallization and chromatography.
L-Ascorbic acid can be produced from L-sorbose or D-sorbitol by using a combination of organisms, one organism having the ability to convert 2-keto-L-gulonic acid to L-ascorbic acid, or 2-keto-D-gluconic acid to D-erythorbic acid, the other organism having L-sorbose/L-sorbosone dehydrogenase and D-sorbitol dehydrogenase, and the ability to convert D-sorbitol and/or L-sorbose to 2-keto L-gulonic acid (see A. Fujiwara et al., EP 213 591; T. Hoshino et al., U.S. Pat. No. 4,960,695; T. Hoshino et al, U.S. Pat. No. 5,312,741), such as, for example, Gluconobacter oxydans DSM 4025 in a one-step conversion with one vessel, or a two-step conversion with two vessels.
D-Erythorbic acid can be produced from D-glucose or D-gluconic acid by using a combination of organisms, one having the ability to convert 2-keto-L-gulonic acid to L-ascorbic acid, or 2-keto-D-gluconic acid to D-erythorbic acid, the other having D-glucose dehydrogenase (Ameyama et al., Agric Biol. Chem. 45:851-861, 1981) and/or D-gluconate dehydrogenase (Shinagawa et al., Agric Biol. Chem. 48: 1517-1522, 1984), such as, for example, Gluconobacter dioxyacetonicus IFO 3271, which can convert D-glucose and/or D-gluconic acid to 2-keto-D-gluconic acid in a one-step conversion with one vessel, or a two step-conversion with two vessels.
The following Examples are provided to further illustrate the process of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.